Methods for identifying inhibitors of botulinum neurotoxins

ABSTRACT

A system and method for identifying a  botulinum  neurotoxin inhibitor employing a  botulinum  neurotoxin substrate complex having a peptide substrate, preferably SNAP-25, a reporter domain on one side of said peptide substrate and an immobilization domain on the opposite side of said peptide substrate. The  botulinum  neurotoxin inhibitor is identified by its ability to decrease the relative amount of cleaved complex, detected through measuring a decrease in complex bound to a solid support. The method of the present invention also utilizes novel cells that express a  botulinum  neurotoxin substrate complex. Also provided are novel stable cell lines that express the  botulinum  toxin substrate complex and viral vectors capable of efficiently expressing an active light chain of the BoNT within mammalian cells.

This application is a continuation and claims priority under 35 U.S.C.§120 from co-pending and co-owned U.S. patent application Ser. No.12/630,336, filed with the United States Patent and Trademark Office onDec. 10, 2009, issued as U.S. Pat. No. 8,093,044 on Jan. 10, 2012, whichis a division U.S. patent application Ser. No. 11/095,055, filed withthe United States Patent and Trademark Office on Mar. 31, 2005, andissued as U.S. Pat. No. 7,632,917B2 on Dec. 15, 2009, which is acontinuation of PCT Application No. PCT/US2003/030899 filed Oct. 3,2003, which, in turn, claims the benefit of U.S. Provisional ApplicationNo. 60/415,177 filed Oct. 1, 2002, all of which are incorporated hereinby reference in their entirety

BACKGROUND

1. Field of Invention

This invention relates to a method for identifying inhibitors ofbotulinum neurotoxins.

2. Background of Invention

Botulinum neurotoxins (BoNT) and tetanus neurotoxin (TeNT) are bacterialproteins that comprise two polypeptide chains connected via a disulfidelinkage. The light chain (˜50 kDa) is disulfide linked to a heavy chain(˜100 kDa). The anaerobic bacterium Clostridium botulinum produces sevenimmunologically distinct but structurally similar neurotoxins designatedBoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F and BoNT/G (collectively,“BoNTs”). After synthesis, highly active neurotoxin is generated byproteolytic cleavage of the clostridial neurotoxins.

These neurotoxins inhibit neurotransmitter release at distinct synapses,which causes two severe neuroparalytic diseases, tetanus and botulism.Many aspects of the cellular and molecular modes of action of thesetoxins have been deciphered. After binding to specific membraneacceptors, BoNTs and TeNT are internalized via endocytosis into nerveterminals. Internalization of toxin is a rapid event and the toxin showspersistent catalytic activity within neurons. Subsequently, the lightchain of the neurotoxin is translocated into the cytosolic compartmentwhere it cleaves one of three essential proteins involved in theexocytotic machinery: (1) synaptosomal associated protein of 25 kDa(SNAP-25); (2) synaptobrevin, also called vesicle associated membraneprotein (VAMP); and (3) syntaxin. Specifically, BoNT/A, BoNT/E andBoNT/C cleave SNAP-25; BoNT/C also cleaves syntaxin. BoNT/B, BoNT/D,BoNT/F, BoNT/G cleave synaptobrevin/VAMP. Tetanus neurotoxin cleavessynaptobrevin/VAMP at the same cleavage site as BoNT/B. See, Schmidt JJ, et al., supra; Anne C, et al., Anal Biochem (2001 291:253-61).

The location of the enzymatic subunit of the clostridial neurotoxins hasbeen mapped to the light chain, which has Zn endopeptidase activity. Thebinding and translocation motifs in a BoNT are located within the heavy(H) chain. All of the BoNT serotypes bind to receptors/acceptors on thepresynaptic terminals of motor neurons at the neuromuscular junction.Schiavo G, et al., (1993) FEBS Lett 335:99-103. The binding of the BoNTto the presynaptic terminal is mediated by the C-terminal domain of theheavy chain (HC) of the toxin. Schiavo G, et al., J Biol Chem (1993)268: 23784-7 and Schiavo G, et al., Nature (1992) 359: 832-5. Binding isfollowed by endocytosis of the toxin into vesicles at the presynapticterminal. As the endocytotic vesicle is acidified, the N-terminus of theHC forms a pore in the vesicle membrane. The light chain (LC)disassociates from HC to act as a zinc-dependent protease that cleavesand inactivates SNARE proteins essential for exocytosis ofneurotransmitter. Amon S S, et al., JAMA 2001, 285:1059-70. In the caseof BoNT/A (the most potent and persistent of the BoNTs) the substrate isSNAP-25, a SNARE protein which resides on the cytoplasmic surface of thepresynaptic membrane. See, Foran P, et al., Biochemistry (1996)35:2630-6; Lewis J, et al., Nat Med (1999) 5:832-5; and Schmidt J J, etal., Anal Biochem (2001) 296:130-7.

The botulinum neurotoxin cleaves the substrate proteins at highlyspecific sites. BoNT/A cleaves SNAP-25 at residues 197/198 (amino acidsQR). See, Foran P, et al., Biochemistry (1996) 35:2630-6; and Lewis J,et al., (1999) supra. BoNT/E cleaves SNAP-25 at residues 180/181 (aminoacids RI).

The unique specificities of BoNT/A and BoNT/E for SNAP-25 was suggestedto be directed through the recognition of a nine residue sequence,termed the SNARE motif. The SNARE motif is about 50 amino acids inlength and assumes a coiled confirmation. The SNARE motif in SNAP-25 iscommon to the other two SNARE proteins: VAMP and syntaxin. SNAP-25, VAMPand syntaxin are the only known substrates of the seven clostridialneurotoxins. There are four copies of the SNARE motif present inSNAP-25. Studies on the interaction of SNAP-25 with BoNT/A and BoNT/Eshowed that a single copy of the motif is sufficient for BoNT/A andBoNT/E to recognize SNAP-25. Washbourne P et al., FEBS Lett. (1997)418:1-5. The full kinetic activity of BoNT/A and BoNT/E for SNAP-25requires at least one SNARE motif. Although the copy of the SNARE motifthat is proximal to the SNAP-25 cleavage site is clearly involved inrecognition with BoNT/A and BoNT/E, in its absence, other more distantcopies of the motif are able to support proteolysis. Id.

The proteolytic attack at specific sites in the protein targets forBoNTs and TeNT induces perturbations of the fusogenic SNARE complexdynamics. These alterations can account for the inhibition ofspontaneous and evoked quantal neurotransmitter release caused by theneurotoxins.

The botulinum neurotoxins (BoNTs) are some of the most potent andpersistent toxins known and can be delivered by an oral or inhalationroute. These properties have contributed to attempts by others to useBoNT as a bioweapon. No effective antidote for BoNT intoxication isavailable. Current therapy consists primarily of long term ventilatorsupport, although early administration of hyperimmune antiserum withinthe first 12 hours can shorten the duration of paralysis. This therapycurrently involves administration of horse serum derived antibodies withthe risks of anaphylactic reaction. Human hyperimmune antiserum is usedto treat infantile botulism. Human hyperimmune antiserum is too limiteda source for use in a bioterrorism attack involving BoNT. Monoclonal IgGantitoxins are being pursued for BoNT therapy, but at least threedifferent monoclonal antibodies are required to inhibit each of theserotypes of botulinum neurotoxin. The cost of producing an oligoclonaltreatment consisting of 15-18 monoclonal antibodies is not commerciallyfeasible.

Immunization is currently the major biodefense strategy against BoNTattacks. Although vaccination can clearly protect against the paralyticeffects of the toxin, there are clear limitations to this strategy whichinclude: 1) the need to vaccinate a large at risk population to preventdisease in even a small number of exposed individuals; 2) activevaccination must be accomplished well before exposure to the toxin; 3)strains of BoNT can be engineered for bioterrorism, that can evadeimmune defense or delivered by viral vector overcoming host immunity(See Fishman P S, et al., Nat Toxins 1999, 7:151-6), and; 4) vaccinationwill interfere with the potential future use of BoNT for medicalconditions and deny the current standard of care to immunized patients.Oyler G A, et al., IBRCC (2001).

An alternative strategy to vaccination against BoNT is the developmentof a clinically useful antidote. Oyler G A, et al., InteragencyBotulinum Research Coordinating Committee, 2001. This strategy opens awide array of possibilities based on the understanding of the molecularpathogenesis of intoxication.

Methods to detect botulinum neurotoxin's catalytic activity have beenbased on detecting SNARE protein cleavage products in vitro. See, forexample, Schmidt U.S. Pat. No. 5,965,699, the contents of which arehereby incorporated by reference in their entirety.

The blocking proteolytic activity of the catalytic light chain is acandidate for treatments to inhibit and terminate the action of thetoxin. SNARE protein cleavage is a late event in intoxication.

Rapid replenishment of SNARE proteins normally occurs and could resultin early restoration of neuromuscular synaptic function Inhibitors thatare able to reach the site of action in the cytosolic compartment of thepre-synaptic terminal of the neuromuscular junction (unprotected by theblood-brain/nerve-barrier) could decrease the neurotoxin's effect ininfected individuals. There is a need for a method to identify aclinically relevant botulinum catalytic inhibitor that penetrates to theintracellular site of action of the toxin and is non-toxic to livingcells. Therefore, a need exists for a method for screening inhibitors ofbotulinum neurotoxin type A (BoNT/A), to identify neurotoxin inhibitorsthat function both in vitro and in living cells. There is also a needfor a method of screening inhibitors of botulinum neurotoxin type E(BoNT/E), type C (BoNT/C), type B (BoNT/B), type D (BoNT/D), type F(BoNT/F) and type G (BoNT/G) that can be used to identify neurotoxininhibitors that function both in vitro and in living cells. In order tofacilitate the identification and development of such botulinum toxininhibitors, there is a need for a system to rapidly assess botulinumtoxin catalytic activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a design of an in-vitro assay for BoNT catalytic activity. Thein-vitro assay for BoNT activity based on separation of a reporterdomain and immobilization domain upon cleavage of SNAP-25 by BoNT.Cleavage of YFP-SNAP-25-His×6 immobilized on metal ion resin by BoNTreleases yellow fluorescent protein (YFP) into the supernatant, whichcan be monitored by YFP fluorescence.

FIG. 2 is comprised of FIGS. 2A, 2B, 2C, 2D, 2E, and 2F. Cleavage ofYFP-SNAP-25-His×6 by BoNT/A and E, compared to a Control (C) is shown inFIG. 2E. GST-SNAP-25, GST-SNAP-25-His×6 and YFP-SNAP-25-His×6 wereefficiently cleaved by BoNT/A and E in vitro (FIGS. 2A and 2D,respectively). GST-SNAP-25 (1-197) and GST-SNAP-25 (1-180) arerecombinant proteins corresponding to the cleaved fragments from BoNT/Aand E cleavage, respectively (FIGS. 2B and 2C). GST-SNAP-25 A/NC, whichharbors a single point mutation (R198T) that renders it BoNT/Aresistant, was cleaved only by BoNT/E in this assay (FIG. 2F).

FIGS. 3A and 3B show the results of the in-vitro assay for BoNTcatalytic activity. YFP-SNAP-25-His×6 immobilized on Nickel resin wereincubated with BoNT/A at 37° C. for 4 hours without agitation. Theamount of YFP fluorescence released into the supernatant was monitoredwith a fluorescence plate reader. The assay was sensitive enough todetect 0.1 U/mL and 1.0 U/mL (1.0 ng/mL and 5.0 ng/mL) of BoNT/A.

FIG. 4 is comprised of FIGS. 4A and 4B. FIG. 4A shows the results of aassays in which synthetic BoNT/A LC is expressed in mammalian cells andcatalytically active. In FIG. 4A, mouse brain extract was incubated withlysates prepared from HEK 293 cells that were transiently transfectedwith BoNT/A LC. Immunoblots showed that mouse endogenous SNAP-25 wascleaved by the BoNT/A LC expressed in HEK 293 cells. FIG. 4B shows theresults of assays employing primary neuronal cultures and HEK 293 cellsstably expressing YFP-SNAP-25-His×6. Both types of cell cultures wereinfected with a Sindbis virus overexpressing BoNT/A LC (moi 5) andanalyzed for SNAP-25 cleavage by immunoblot. The synthetic BoNT/A LC wasefficiently expressed and cleaved both endogenous neuronal SNAP-25 andYFP-SNAP-25-His×6.

FIG. 5 is a photomicrograph of HEK 293 cells stably expressingYFP-SNAP-25-His×6. Cells were imaged for YFP fluorescence which showedthe proper localization of YFP-SNAP-25-His×6 at the cell membranes.

FIG. 6 shows is a schematic illustration of the cell-based assay forBoNT catalytic activity. HEK 293 cells stably expressingYFP-SNAP-25-His×6 are exposed to BoNT and the amount ofYFP-SNAP-25-His×6 cleavage monitored by the quantity of YFP fluorescencebound to Nickel resin. A high-throughput cell-based assay uses a similarassay platform to that used in the in-vitro assay, to monitor theneurotoxin proteolytic activity on a substrate located in cells.

FIG. 7 shows the results of a cell-based assay for BoNT catalyticactivity in which HEK 293 cell-lines YSH5b and YSH12b were infected withSindbis virus over-expressing recombinant BoNT/A LC. The expressedBoNT/A LC efficiently cleaves YFP-SNAP-25-His×6 in these cells,resulting in a decrease in YFP fluorescence reporter signal bound aNickel column.

FIGS. 8A and 8B are the synthetic BoNT/A and BoNT/E sequences,respectively in which the BamHI and AccIII restriction enzymes sites areidentified.

DETAILED DESCRIPTION OF THE INVENTION

This present invention is the first system for screening inhibitors ofbotulinum neurotoxin type A (BoNT/A) for use in both in vitro and inliving cells. Such a system can be used to greatly accelerate the searchfor a clinically useful antidote to botulism.

All references cited herein are hereby incorporated by reference intheir entirety.

This is a novel system for monitoring the catalytic activity of a BoNTboth in vitro and within living cells. The system is designed tofacilitate the identification of clinically useful antidotes forbotulinum neurotoxin type A and can be adapted for use as a highthroughput screening assay system.

The system of the present invention provides a method for detecting BoNTactivity and identifying inhibitors of BoNT activity by monitoring thecleavage of the neurotoxin's endogenous substrate using a novelrecombinant protein, referred to as a botulinum neurotoxin substratecomplex (or substrate indicator protein). The botulinum neurotoxinsubstrate complex of the present invention is comprised of: (a) apeptide substrate that is capable of being cleaved at a specificcleavage site by a botulinum neurotoxin; (b) a reporter domain on oneside of the peptide substrate; and (c) an immobilization domain on theopposite side of the peptide substrate. The preferred peptide substratesare SNAP-25, a SNAP-25 isoform, syntaxin, a syntaxin isoform, VAMP, aVAMP isoform, and peptides having at least 80% identity to the foregoingas long as the peptide substrate is capable of being cleaved at aspecific cleavage site by a botulinum neurotoxin. The more preferredpeptide subtrates are SNAP-25, syntaxin and VAMP. The most preferredpeptide substrate is SNAP-25 because it is the endogenous substrate ofBoNT/A and BoNT/E, which are the two serotypes that account for themajority of botulinum infections. The nucleotide and amino acid sequenceencoding murine SNAP-25 is shown in SEQ ID No. 15 and SEQ ID No. 16,respectively.

The system of the present invention for detecting BoNT/A or BoNT/Eactivity and identifying inhibitors of BoNT/A or BoNT/E activity isbased on methods for monitoring the cleavage of their endogenoussubstrate SNAP-25. The system of the present invention monitors theproteolytic cleavage of SNAP-25 using a novel recombinant protein,referred to as a botulinum neurotoxin substrate complex. In oneembodiment of the SNAP-25 botulinum neurotoxin substrate complex(YFP-SNAP-25-His×6), the complex is comprised of the protein substrateSNAP-25, which has a hexahistidine peptide (His×6) immobilization domainat its carboxyl terminus and a yellow fluorescent protein (YFP) reporterdomain at its amino-terminus. The YFP-SNAP-25-His×6 example of abotulinum neurotoxin substrate complex is illustrated graphically inFIG. 1. This YFP-SNAP-25-His×6 system can also be used to detect BoNT/Cactivity and identify inhibitors of BoNT/C.

The YFP-SNAP-25-His×6 complex is capable of binding to nickel resinbeads through its C-terminal His×6 immobilization domain of the complex.Nickel resin coated 96 well microtiter plates are suitable for highthroughput screening and are commercially available (Pierce). TheYFP-SNAP-25-His×6 complex is bound to the nickel resin in the wells ofthe plate. In the presence of BoNT/A, BoNT/C, or BoNT/E, the complex iscleaved to produce a cleaved complex, liberating the fluorescentindicator YFP reporter domain into the supernatant and leaving theimmobilized domain bound to the Nickel. The remaining intact complex(containing the reporter domain) present on the plates and/or thereporter domain released into the supernatant can be monitored; the YFPreporter is monitored by YFP fluorescence. There is an inversecorrelation between toxin concentration in the well and YFP fluorescencebound to plate. In other words, the greater the concentration of BoNT,the lower the concentration of YFP-SNAP-25-His×6 complex bound to theplate because the toxin releases the YFP reporter domain from the plate.

This approach confers a substantial advantage over other BoNT assays bycapturing the “free” (proteolytically-liberated) portion containingfluorescence, enzyme activity or other detection signature. Thisstrategy improves assay sensitivity and reduces background, thuspermitting even very low amounts of the (proteolyzed) product to bemeasured. The complex is immobilized on a nickel surface through aC-terminal hexahistadine immobilization domain. This approacheffectively removes unwanted background materials from the test sampleand permits the reduction in bound reporter domain in the immobilizedcomplex to be measured.

The system of the present invention further provides methods adapted forcell based screening to monitor the catalytic activity of a BoNT inliving cells and to identify molecules that inhibit the catalyticactivity of a BoNT in living cells. The present invention provides novelstable cell lines that express the botulinum toxin substrate complex(e.g., YFP-SNAP25-His×6 or GST-SNAP25-His×6). In one embodiment of thepresent invention, a viral vector capable of efficiently expressing anactive light chain of BoNT within mammalian cells is provided.

Both the botulinum toxin substrate complex component and the BoNTexpressing viral vector component of the system are suitable for use inhigh throughput methods. Commercially available multi-titer platescoated with nickel resin are capable of binding to the substrateindicator protein (i.e., the neurotoxin substrate complex) of thepresent invention. Stable YFP-SNAP-25-His×6 expressing cell lines willgrow consistently within multi-titer plates as well. Plates of such celllines allow for simultaneous, consistent infection with Sindbis virusexpressing the synthetic BoNT LC in all wells. These dually expressingcells create multiple replicates per plate, where each well is availableas a test vessel for a putative BoNT inhibitor. Also, lysates from suchcells can be incubated and washed in the resin coated wells and theplates can be assessed for bound YFP fluorescence using a multi-wellfluourimeter. Libraries of compounds, having established or potentialinhibitory properties for metal protease, can be screened for theirpotency as a BoNT inhibitor. A compound identified as a BoNT inhibitorcan be developed for use as a BoNT antidote.

EXAMPLE 1 Construction and Expression of YFP-SNAP-25-His×6

To construct YFP-SNAP25-His×6, PCR is used to generate His×6 tag(histidine tag) at the carboxyl terminus of mouse SNAP-25 (shown in SEQID No. 16) and ligated into EYFP-C1 vector (Clontech). Similarconstructs can be made with GST reporter domain encoding vectors. Forbacterial expression, PCR is used to generate YFP-SNAP25-His×6 withappropriate restriction sites and cloned into pGEX4T3 vector (AmershamPharmacia Biotech). The protein is expressed in BL21(DE3) (Stratagene)bacteria and purified with glutathione sepharose 4B (AP Biotech) and theGST motif removed with thrombin cleavage. YFP-SNAP-25-His×6 is alsocloned into pET vector (Novagen), expressed in BL21(DE3) bacteria andpurified by nickel affinity chromatography. The in-vitro assay describedbelow uses the protein purified from pGEX vector.

EXAMPLE 2 Generation of YFP-SNAP-25-His×6 Cell-Lines

HEK 293 cells were cultured in Minimum Essential Medium (GIBCO)supplemented with 10% fetal bovine serum, L-glutamine (10%) andpen/strep antibiotics (1%). HEK 293 cells were transfected withYFP-SNAP-25-His×6 plasmid and cultured in media containing G418 untilisolated foci emerged. Isolated foci were selected for expansion andscreened by immunoblots to obtain clonal cell-lines stably expressingYFP-SNAP-25-His×6.

EXAMPLE 3 Viral RNA Transcription, Transfection and Plaque Assays

Purified plasmid DNAs were linearized by digestion with XhoI andtranscribed using SP6 polymerase in the presence of cap analog.Transcription reactions were used for transfection of BHK-21 cells usingstandard methods. BHK-21 cells (ATCC) were cultured in Dubelco's MinimumEssential Medium (GIBCO) supplemented with 10% fetal bovine serum,L-glutamine (10%) and pen/strep antibiotics (1%). Plaque formation wasassayed using BHK-21 monolayers.

EXAMPLE 4 In Vitro Assay for BoNT Catalytic Activity

The catalytic activity of BoNT/A was assayed using YFP-SNAP-25-His×6immobilized on Nickel resin in 96-well plates. PurifiedYFP-SNAP-25-His×6 protein was immobilized on the resin and washedextensively in PBS. BoNT/A was added in the range of 1-100 U/mL PBS. Theplates were incubated at 37° C. without agitation. The reaction wasquenched with EDTA and the supernatant monitored for YFP fluorescence ina fluorescence plate reader.

The basis for the detection of BoNT A or E activity is the cleavage ofSNAP-25. Cleavage is monitored with a novel recombinant protein whereSNAP-25 has a hexahistidine peptide (“His×6” or “histadine tag”) fusedto its carboxyl terminus and the yellow fluorescent protein (YFP) fusedto its amino-terminus (YFP-SNAP-25-His×6, FIG. 1). The histadine tagmolecule binds to nickel resin beads through its C-terminal His×6. Suchnickel resin is bound to 96 well microtiter plates, which arecommercially available (Pierce) and suitable for high throughputscreening. In the presence of BoNT A, C, or E, the boundYFP-SNAP-25-His×6 is cleaved, liberating the fluorescent indicator YFPdomain. There is an inverse correlation between toxin concentration inthe well and YFP fluorescence bound to plate. In other words, thegreater the concentration of toxin, the lower the concentration ofYFP-SNAP-25 bound to the plate because the toxin releases the YFPreporter/signal from the plate.

The YFP-SNAP-25-His×6 immobilized on metal ion resin is cleaved by BoNT,which separates the reporter domain from the immobilization domain andreleases the YFP reporter domain into the supernatant (leaving theimmobilization domain attached to the metal ion resin). The YFP reportercan be monitored by YFP fluorescence.

FIGS. 2A, 2B, 2C, 2D, 2E, and 2F show the results of experimentsconducted to determine if the YFP or GST added to the N-terminus ofSNAP-25 and the charged hexahistidine group at the C-terminus of SNAP-25effects the sensitivity of SNAP-25 to BoNT cleavage. BoNT/A cleavesSNAP-25 only 7 amino-acids from its C-terminus. Each of GST-SNAP-25,GST-SNAP-25-His×6 and YFP-SNAP-25-His×6 were purified from bacterialexpression.

We first demonstrated that BoNT/A and BoNT/E efficiently cleave each ofGST-SNAP-25, GST-SNAP-25-His×6 and YFP-SNAP-25-His×6 in-vitro, as shownin FIGS. 2A, 2D and 2E, respectively. Cleavage of YFP-SNAP-25-His×6 byBoNT/A and E is shown in FIGS. 2A, and FIGS. 2D-2F. GST-SNAP-25,GST-SNAP-25-His×6 and YFP-SNAP-25-His×6 were efficiently cleaved byBoNT/A and E in vitro. GST-SNAP-25 (1-197) and GST-SNAP-25 (1-180) arerecombinant proteins corresponding to the cleaved fragments from BoNT/Aand E cleavage respectively. GST-SNAP-25 A/NC, which harbors a singlepoint mutation (R198T), renders it BoNT/A resistant, was cleaved only byBoNT/E in this assay. GST-SNAP-25 (1-197) is the cleavage fragment fromBoNT/A cleavage of the recombinant protein GST-SNAP-25. GST-SNAP-25(1-180) is the cleavage fragment from BoNT/E cleavage of the recombinantprotein GST-SNAP-25.

A single point mutation in SNAP-25 (R198T) renders SNAP-25 resistant toBoNT/A, but sensitive to cleavage by BoNT/E. In this assay, GST-SNAP-25A/NC is resistant to BoNT/A cleavage, yet sensitive to cleavage byBoNT/E.

A single point mutation in SNAP-25 (D179K) renders SNAP-25 resistant toBoNT/E, but sensitive to cleavage by BoNT/A. An assay employingGST-SNAP-25 (D179K) can be tested to determine if the fusion protein isresistant to BoNT/E cleavage while remaining sensitive to BoNT/A.

A double point mutation in SNAP-25 (D179K and R198T) renders SNAP-25resistant to both BoNT/A and BoNT/E.

To evaluate the sensitivity of an in-vitro assay based on cleavage ofYFP-SNAP-25-His×6, we incubated 96-well plates coated withYFP-SNAP-25-His×6 with 1-100 U/mL BoNT/A and measured the amount offluorescence released into the supernatant. At 4 hours, YFP fluorescencereleased into the supernatant increases almost 10 fold over backgroundin the treated wells. Control wells containing no toxin or varyingquantities from 0.1 U/mL to 100 U/mL (i.e., 1.0 ng/ml to 100.0 ng/ml) ofBoNT/A pre-inactivated by boiling for 5 min show minimal release of YFPfluorescence (FIGS. 3A and 3B). FIGS. 3A and 3B show the in-vitro assayfor BoNT catalytic activity. YFP-SNAP-25-His×6 immobilized on Nickelresin were incubated with BoNT/A at 37° C. for 4 hours withoutagitation. The amount of YFP fluorescence released into the supernatantwas monitored with a fluorescence plate reader. The pilot assay formatwas sensitive enough to detect 0.1 U/mL of BoNT/A. The assay thereforeexhibits sensitivity down to 0.1 U/mL of BoNT/A.

High-throughput screening of inhibitors of BoNT can be achieved byincubating YFP-SNAP-25-His×6 coated plates with BoNT and a putativetoxin inhibitor. Efficacy of any inhibitor of the catalytic activity ofBoNT for SNAP-25 cleavage would be proportional to the increase of boundfluorescence toward that seen in control wells without toxin. Thisapproach confers a substantial advantage over other BoNT assays bycapturing the “free” (proteolytically-liberated) portion containingfluorescence, enzyme activity or other detection signature. Thisstrategy improves assay sensitivity and reduces background, thuspermitting even very low amounts of the (proteolyzed) product to bemeasured.

In one embodiment, intact target protein is immobilized on a nickelsurface through a C-terminal 6× his tag. This approach effectivelyremoves unwanted background materials from the test sample by measuringthe reduction in bound activity in the immobilized complex. The methodof the present invention can measure both the loss of fluorescence fromthe beads as the substrate is cleaved and the increase in freefluorescence in solution. There is also a measurable loss offluorescence from beads. In one embodiment of the high throughput assaymethod of the present invention, Nickel beads are incubated with asolution containing excess GST-YFP-SNAP25-6×His before the beads arewashed in order to load the beads to maximum capacity. The fluorescenceof the loaded beads is measured before they are incubated with Bo/NT andthe fluorescence is measured again. The amount of loss of fluorescenceis proportional to the amount of Bo/NT added. Also the fluorescenceliberated into solution is measure to determine the increase influorescence released into solution.

EXAMPLE 5 Cell-Based Assay for BoNT Catalytic Activity

HEK 293 cells stably expressing YFP-SNAP-25-His×6 were infected withSindbis virus overexpressing catalytic BoNT/A LC at multiplicity ofinfection of 5. At the termination of such a test run, the cells werelysed in a lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl,0.1% NP-40 and protease inhibitors. The lysate was applied to Nickelresin followed by extensive washes in Tris-buffered saline. YFPfluorescence in the flow through or bound to the resin was measuredusing a fluorescence plate reader. This assay can be performed inmulti-well plates and the lysis buffer is added to the wells after theYFP-SNAP-25-His×6 expressing cells are infected with the BoNT/Aexpressing Sindbis virus. The lysated is withdrawn, and applied to areplicate resin coated plate.

Although Sindbis virus is cytopathic, there is a window of at least 24hours from the time of Sindbis virus infection where the stablytransfected HEK 293 express both the recombinant YFP-SNAP-25-His×6 andBoNT/A can be used to test moieties or compounds for their ability toinhibit the toxin's catalytic activity in the cells. In anotherembodiment of the present invention, non-cytopathic forms of the Sindbisvirus can be used to improve cell viability. In yet another embodimentof the present invention, inducible cell-lines that expressYFP-SNAP-25-His×6 and conditionally express the recombinant BoNT LC (inthe presence of an inducer) can be developed and used in the cell-basedassays of the present invention.

The recombinant BoNT/A light chain is efficiently expressed andcatalytically active. To verify that the synthetic BoNT/A light chain LCis catalytically active, HEK 293 cells were transiently transfectedusing a mammalian expression vector containing BoNT/A LC. Incubation ofmouse brain extract with lysates from the transfected cells resulted incleavage of mouse SNAP-25, as monitored by immunoblots (FIG. 4A). FIGS.4A and 4B show that synthetic BoNT/A LC is expressed in mammalian cellsand catalytically active. FIG. 4A shows the results from an assay inwhich mouse brain extract was incubated with lysates prepared from HEK293 cells transiently transfected with BoNT/A LC. Immunoblots showedthat mouse endogenous SNAP-25 was cleaved by BoNT/A LC expressed in HEK293 cells. (B) Primary neuronal cell cultures and HEK 293 cells stablyexpressing YFP-SNAP-25-His×6 were infected with a Sindbis virusoverexpressing BoNT/A LC (moi 5) and analyzed for SNAP-25 cleavage byimmunoblot. The synthetic BoNT/A LC was efficiently expressed andcleaved both endogenous neuronal SNAP-25 and YFP-SNAP-25-His×6.

Infection of primary neuronal cultures with a Sindbis virusoverexpressing the synthetic BoNT/A LC also resulted in efficientcleavage of SNAP-25 in these neurons (FIG. 4B).

Cell-Based Assay for BoNT Catalytic Activity

Any inhibitor of the proteolytic activity of BoNT must have both lowcytotoxicity and high intracellular penetration to be considered as apotential clinical antidote. Toward the goals of identifying clinicallyuseful agents we have developed a cell based system to monitor theproteolytic activity of BoNT/A and BoNT/E. Two different clonal lines ofhuman embryonic kidney cells (HEK293) were produced by transfecting theHEK293 cells so that they express high levels of YFP-SNAP25-His×6. Thesetwo cells lines are identified as YSH5b and YSH12b. HEK 29 cells stablyexpressing YFP-SNAP-25-His×6 are shown in FIG. 5, imaged for YFPfluorescence showing proper localization of YFP-SNAP-25-His×6 to thecell membranes. SNAP-25 is normally associated with cell membranes inneurons. Although HEK 293 cells do not express SNAP-25 endogenously,YFP-SNAP-25-His×6 expressed in these cells was properly localized to thecell membranes. Since HEK 293 cells do not express receptors for BoNT, anovel route is required to intoxicate these cells. We achieve this usinga Sindbis virus vector engineered to express a catalytically active formof the light chain of BoNT (SV-LC).

Both lines of the YFP-SNAP25-His×6 expressing HEK 293 cells (i.e., YSH5band YSH12b) as well as primary dissociated neurons show cleavage ofSNAP-25 when infected with SV-LC (FIG. 4B). Immunoblot analysis of celllysates reveals that all SNAP-25 associated protein (YFP-SNAP25-6His inHEK293 and native SNAP-25 in neurons) from transfected cells has amolecular weight consistent with BoNT/A cleavage.

The cleavage of YFP-SNAP-25-His×6 in the YSH5b and YSH12b cells byexpression of BoNT LC can be monitored quantitatively using the assaysystem that is similar to the in-vitro methods is illustrated in FIG. 6.FIG. 6 illustrates one embodiment of the cell-based assay for BoNTcatalytic activity. HEK 293 cells stably expressing YFP-SNAP-25-His×6are exposed to BoNT and the amount of YFP-SNAP-25-His×6 cleavage ismonitored by the YFP fluorescence bound to Nickel resin. This allow forhigh-throughput cell-based assay using similar assay platform as thein-vitro assay.

Cleavage of the recombinant YFP-SNAP-25-6His by BoNT LC produces celllysates containing YFP-SNAP-25 devoid of the His tag, which thereforeresults in a reduced quantity of fluorescence bound to nickel resinwells (FIG. 7). FIG. 7 shows the results from a cell-based assay forBoNT catalytic activity. HEK 293 cell-lines YSH5b and YSH12b wereinfected with Sindbis virus over-expressing recombinant BoNT/A LC. Theexpressed BoNT/A LC efficiently cleaves YFP-SNAP-25-His×6 in these cellsresulting in a decrease in YFP fluorescence bound the Nickel column. Ifa molecule can enter the cells and inhibit activity of the BoNT lightchain, resin bound fluorescence will be restored to control (non-SVLCinfected) levels.

EXAMPLE 6 Construction of Type A and Type E BoNT Light Chains

Clostridial genes are aberrantly A/T rich and poorly translated ineukaryotic cells. To achieve efficient expression of the BoNT LC, wereconstructed codon-substituted BoNT/A and BoNT/E LC with thesecriteria: (1) preferred codon usage in E. coli and eukaryotic cells, (2)divide the LC into interchangeable domains to facilitate the design ofchimeric BoNT LC, (3) insert restriction sites compatible with severaltypes of expression systems. BoNT/A and /E LC are constructed in PCRreactions by overlap extension of oligonucleotides as building blocks.The synthetic LC are subcloned into appropriate mammalian expressionvector and Sindbis virus vector.

Based on these criteria, synthetic BoNT/A and BoNT/E LCs were designed,introducing internal BaM H1 and Acc III sites into the gene to createmodules “1”, “2”, and “3” (5′ to 3′). The synthetic genes wereengineered to include tandem Xho I, Nhe I, and Sph I sites at their 5″ends and Apa I on the 3′ end. The sequence of the synthetic BoNT/A LCgene (SEQ ID No. 1) is shown in FIG. 8A and sequence of the syntheticBoNT/E LC gene (SEQ ID No. 2) is shown in FIG. 8B. FIG. 8A shows thenucleotide sequence (SEQ ID NO: 1) encoding BoNT/A LC optimized forexpression in eukaryotic cells in which a BamHI (bold and underlined)and AccIII (italics+double underlined) restriction enzyme sites havebeen engineered. FIG. 8B shows the nucleotide sequence (SEQ ID NO: 2)encoding BoNT/E LC optimized for expression in eukaryotic cells in whicha BamHI (bold and underlined) and AccIII (italics+double underlined)restriction enzyme sites have been engineered.

Oligonucleotides of 50-60 nt were designed in pairs to introduceoverlapping regions of 12 nt at their opposing ends. The oligos wereoptimized for preferred codon usage in E. coli and eukaryotic cells.These pairs were extended and amplified by using PCR to create fragmentsof ˜100 nt, which were then utilized as building blocks in successiverounds of PCR with oligos having 12 nt overlaps with the ends of theprior PCR amplification. This type of “overlapping PCR” gene synthesiswas utilized to create the entire synthetic gene. To monitor thefidelity of the gene construction process, PCR fragments were clonedinto TA TOPO cloning vectors (Invitrogen) at regular intervals andsequenced to obtain template lacking mutations in coding sequence orrestrictions sites.

The light chain sequences for BoNT/A and BoNT/E were divided into threesections by creating the internal restriction sites, BamHI (GGATCC(underlined in the sequences above) and AccIII (TCCGGA (doubleunderlined in the sequences above) sites, without changing the aminoacid sequence of the light chains (silent mutagenesis). For eachserotype, Fragment One is from the ATC codon to the BamHI site(underlined). Fragment Two is from the BamHI site to the AccIII site(double underlined). Fragment Three is from the AccIII site to the finalCAT codon.

Synthetic genes for the BoNT/A and BoNT/E LCs were subcloned intoappropriate expression systems for biological applications. For thosetransient recombinant protein expression applications requiring plasmidthe completed recombinant genes were transferred to pcDNA 3.1, usingunique NheI and ApaI restriction sites in the expression plasmid in thefollowing manner: (a) Fragment One is excised using SpeI/BamHI andligated into NheI/BamHI sites of pcDNA3.1(+); (b) Fragment Three is cutwith SpeI/ApaI and ligated into the XbaI/ApaI sites ofpcDNA3.1(+)/Fragment 1; (c) finally, Fragment Two is cloned in usingBamHI/AccIII to get the complete light chain sequence. Afterverification by DNA sequencing of the insert ligation sites, preparativeamounts of plasmid were purified.

EXAMPLE 7 Construction of YFP-BoNT/A and YFP-BoNT/E Expressing Vectors

For recombinant protein expression applications requiring introductionof mammalian virus the genes were transferred to pSindREP 5(Invitrogen), using unique Xba I and Apa I sites in the viral DNA vector(note: Xba I and Nhe I restriction sites have compatible ends forligation) (NheI/ApaI sites for the insert). pVSindREP5 can be used tomake viral replicons (ie. replication-deficient virus). Afterverification by DNA sequencing of the insert ligation sites, preparativeamounts of the DNAs were purified for later expression studies. Forstudies of expression using fluorescent fusion proteins, the BoNT/A andBoNT/E LC genes were transferred to a plasmid containing the codingsequence for yellow fluorescent protein (YFP) pEYFP (EYFP-C1 fromClontech), using unique Xho I and Apa I sites. After verification by DNAsequencing of the insert ligation sites, preparative amounts of plasmidwere purified.

YFP-BoNT/A and YFP-BoNT/E expression vectors are also constructed byligating the synthetic light chains into the EYFP-C1 vector (Clontech)using XhoI/ApaI sites to facilitate tracking the light chains inseparate experiments.

EXAMPLE 8 Construction of Sinbis Viral Vector for Expressing BoNT LightChains

For recombinant protein expression applications requiring introductionof mammalian virus, the genes are transferred to Sindbis virus vectorpVSind 1 using XbaI/NotI sites in the vector (NheI/NotI in the insert).pVSind 1 vector is modified from the TE12Q strain of Sindbis describedby Lewis J, et al., Nat Med (1999) 5:832-5, which is hereby incorporatedby reference in its entirety. This construct is used to makereplication-competent Sindbis virus.

EXAMPLE 9 Construction of pGEX6P2 Vector for Expressing BoNT LightChains

The BoNT/A is also cloned into pGEX6P2 vector (Amersham PharmaciaBiotech) to express in bacteria as a source of BoNT/A light chain forantibody production and in-vitro assays, so that the risk associatedwith using the holotoxin can be minimized.

EXAMPLE 10 Construction of Chimeric BoNT/A and BoNT/E LCs.

The addition of the unique restriction sites within the BoNT/A andBoNT/E light chains described herein also allows for convenient swappingof domains from BoNT/A and E light chains for creation of chimeric lightchains in order to produce light chains having novel properties for usein identifying inhibitors of BoNTs and for use themselves as therapeuticproducts.

The intermediate constructs are cloned into TA TOPO cloning vectors(Invitrogen) to check for PCR fidelity by sequencing. The completedfragments of the sequence are then ligated using the internalrestriction sites in pcDNA3.1(Neo+): (a) Fragment One is excised usingSpeI/BamHI and ligated into NheI/BamHI sites of pcDNA3.1(+); (b)Fragment Three is cut with SpeI/ApaI and ligated into the XbaI/ApaIsites of pcDNA3.1(+)/Fragment 1; (c) finally, Fragment Two is cloned inusing BamHI/AccIII to product a complete chimeric light chain sequence.The chimeric light chains are constructed by the same procedure withdifferent combinations of Fragments One, Two, Three of BoNT/A andBoNT/E. After each assembly step, verification of correct ligation wascarried out by DNA sequencing. The following chimeric LCs weretransferred to pCDNA 3.1, using the unique Nhe I and Apa I sites in theexpression plasmid: 1) A1-A2-E3, 2) A1-E2-E3, 3) A1-E2-A3, 4) E1 -A2-A3,5) E1-A2-E3, 6) E1-E2-A3. These same chimeric LCs were transferred tothe replication-competent Sindbis expression vector pVSind1, usingunique Xba 1 and Not 1 sites, the latter derived from the TA vector. Inaddition to the chimeras, the full-length BoNT/A and BoNT/E LC geneswere transferred to the pVSind1 vector to enable comparison with thechimeric forms.

Alternative Embodiments

One can easily use green fluorescent protein (GFP) instead of yellowfluorescent protein (YFP). Furthermore, reporter moieties other thanfluorescent markets can be used. For example, colorimetric substratereactions such as beta-galactosidase, alkaline phosphatase, orglutathione-S-transferase (GST) or other enzymes along with theappropriate substrate or antibody (for an immunoassay) can be used. Anabsorption assay can be used to detect inhibitors of BoNT activity. Someexamples of other enzymes and substrates can be found in U.S. Pat. No.6,197,534. Any reporter compound which can be detected in animmunoassay, absorption assay, or substrate assay can be used.

A preferred embodiment is described as an indicator for BoNT/A and as anindicator for BoNT/E. The present invention can also be easily adaptedby those of skill in the art for monitoring syntaxin cleaving by BoNT/Cor VAMP/synaptobrevin cleaving by BoNT/B, BoNT/D, BoNT/F, and BoNT/G.

The nucleic acid and amino acid sequences referenced in the instantspecification can be found in the corresponding SEQ ID Numbers, whichare identified in Table 1 below, the sequence listing of each of whichis hereby incorporated by reference in its entirety.

TABLE 1 SEQ ID No. Type of Sequence and Protein Encoded 1 BoNT/A nucleicacid 2 BoNT/E nucleic acid 3 BoNT/A amino acid 4 BoNT/E amino acid 5BoNT/C nucleic acid 6 BoNT/C amino acid 7 BoNT/B nucleic acid 8 BoNT/Bamino acid 9 BoNT/D nucleic acid 10 BoNT/D amino acid 11 BoNT/F nucleicacid 12 BoNT/F amino acid 13 BoNT/G nucleic acid 14 BoNT/G amino acid 15Murine SNAP-25 nucleic acid 16 Murine SNAP-25 amino acid

In describing representative embodiments of the invention, thespecification may have presented the method and/or process of theinvention as a particular sequence of steps. However, to the extent thatthe method or process does not rely on the particular order of steps setforth herein, the method or process should not be limited to theparticular sequence of steps described. As one of ordinary skill in theart would appreciate, other sequences of steps may be possible. Inaddition, the claims directed to the method and/or process of theinvention should not be limited to the performance of their steps in theorder written, to the extent that the method or process does not rely onthe particular order of steps, and one skilled in the art can readilyappreciate that the sequences may be varied and still remain within thespirit and scope of the invention.

The foregoing disclosure of the embodiments of the invention has beenpresented for purposes of illustration and description. It is notintended to be exhaustive or to limit the invention to the precise formsdisclosed. Many variations and modifications of the embodimentsdescribed herein will be apparent to one of ordinary skill in the art inlight of the above disclosure.

REFERENCES

-   1. Adler M., et al.,. Pharmacological countermeasures for Botulinum    intoxication. (Chapter 12) Advances in low dose exposure to chemical    and biological weapons. CRC Press. 2001. pp 373-387.-   2. Amon S S, et al.,. Working group on Civilian Biodefense.    Botulinum toxin as a biological weapon: medical and public health    management. JAMA 2001, 285:1059-70.-   3. Marks J D. Advances in monoclonal antibody treatment of BoNT    intoxication. Interagency Botulinum Research Coordinating Committee.    October 2001. Abstract.-   4. Cohen J, et al., Bioterrorism. Vaccines for biodefense: a system    in distress. Science 2001, 294:498-501.-   5. Oyler G A, et al., Development of a cell-based high-throughput    screening system for inhibitors of Botulinum toxin. Interagency    Botulinum Research Coordinating Committee, October 2001.-   6. Simpson L L. Botulinum toxin and tetanus toxin recognize similar    membrane determinants. Brain Res 1984, 305: 177-80.-   7. Lalli G, et al., Functional characterization of tetanus and    Botulinum neurotoxins binding domains. J Cell Sci 1999, 112:    2715-24.-   8. Oyler G A, et al., The identification of a novel    synaptosomal-associated protein, SNAP-25, differentially expressed    by neuronal subpopulations. J Cell Biol 1989, 109:3039-52.-   9. Blasi J, et al., Botulinum neurotoxin A selectively cleaves the    synaptic protein SNAP-25. Nature 1993, 365: 160-3.-   10. Schiavo G, et al., Botulinum neurotoxins serotypes A and E    cleave SNAP-25 at distinct COOH-terminal peptide bonds. FEBS Lett    1993, 335:99-103.-   11. Schiavo G, et al., Identification of the nerve terminal targets    of Botulinum neurotoxin serotypes A, D and E. J Biol Chem 1993: 268:    23784-7.-   12. Schiavo G, et al., Tetanus and Botulinum-B neurotoxins block    neurotransmitter release by proteolytic cleavage of synaptobrevin.    Nature 1992, 359: 832-5.-   13. Schiavo G, et al., C. Botulinum neurotoxin serotype F is a zinc    endopeptidase specific for VAMP/synaptobrevin. J Biol Chem 1993,    268: 11516-9.-   14. Yamasaki S, et al., Botulinum neurotoxin serptype G proteolyses    the Ala81-Ala82 bond of rat synaptobrevin 2. Biochem Biophys Res    Comm 1994, 200: 829-35.-   15. Lewis J, et al., Inhibition of virus-induced neuronal apoptosis    by Bax. Nat Med 1999, 5:832-5.-   16. Schmidt J J, et al., High-throughput assays for botulinum    neurotoxin proteolytic activity: serotypes A, B, D, and F. Anal    Biochem 2001 Sep. 1; 296(1):130-7.-   17. Anne C, et al., High-throughput fluorogenic assay for    determination of Botulinum type B neurotoxin protease activity. Anal    Biochem 2001, 291: 253-61.-   18. Agapov E V, et al., Noncytopathic Sindbis virus RNA vectors for    heterologous gene expression. Proc Natl Acad Sci USA 1998 Oct. 27,    95(22):12989-94.

1. A method for identifying a botulinum neurotoxin inhibitor comprisingthe steps of: (i) exposing botulinum neurotoxin substrate complexexpressing cells to a botulinum neurotoxin, in the presence and absenceof a test molecule, wherein said botulinum neurotoxin substrate complexcomprises: (a) a peptide substrate selected from the group consisting ofsynaptosomal associated protein of 25 kD (SNAP-25), a SNAP-25 isoform,syntaxin, a syntaxin isoform, vesicle-associated membrane protein(VAMP), a VAMP isoform, and peptides having at least 80% identity to theforegoing, wherein said peptide substrate comprises a motif of solubleNSF (N-ethylmaleimide-sensitive fusion protein) attachment proteinreceptor (a SNARE motif); (b) a reporter domain comprising a fluorescentprotein covalently attached to one side of said peptide substrate; and(c) an immobilization domain covalently attached to an opposite side ofsaid peptide substrate, wherein said complex is cleaved to produce acleaved complex in the absence of said test molecule; (ii) lysing saidcells and collecting cell lysate from said cells; (iii)immobilizing thesubstrate complex on a solid support; (iii) comparing the effect of thepresence of said test molecule on the amount of said cleaved complex tothe amount of said cleaved complex in the absence of said test molecule;and (iv) identifying the test molecule as a botulinum neurotoxininhibitor if the presence of said test molecule decreases the relativeamount of cleaved complex produced in said cells as compared to theamount of cleaved complex produced in said cells in the absence of saidtest molecule; wherein said botulinum neurotoxin in step (i) isdelivered to said cells by means selected from administering a botulinumneurotoxin to said cells and expressing said botulinum neurotoxin insaid cells by a recombinant vector; and wherein said recombinant vectorcomprises a nucleic acid sequence that is from about 80% to 100%homologous to the nucleic acid sequences represented in the groupconsisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13, wherein said sequenceencodes a neurotoxin that is capable of cleaving the specific cleavagesite of the endogenous substrate of said neurotoxin. A method foridentifying a botulinum neurotoxin inhibitor comprising the steps of:(i) exposing botulinum neurotoxin substrate complex expressing cells toa botulinum neurotoxin, in the presence and absence of a test molecule,wherein said botulinum neurotoxin substrate complex comprises: (a) apeptide substrate selected from the group consisting of synaptosomalassociated protein of 25 kD (SNAP-25), a SNAP-25 isoform, syntaxin, asyntaxin isoform, vesicle-associated membrane protein (VAMP), a VAMPisoform, and peptides having at least 80% identity to the foregoing,wherein said peptide substrate comprises a motif of soluble NSF(N-ethylmaleimide-sensitive fusion protein) attachment protein receptor(a SNARE motif); (b) a reporter domain comprising a fluorescent proteincovalently attached to one side of said peptide substrate; and (c) animmobilization domain covalently attached to an opposite side of saidpeptide substrate, wherein said complex is cleaved to produce a cleavedcomplex in the absence of said test molecule; (ii) lysing said cells andcollecting cell lysate from said cells; (iii)immobilizing the substratecomplex on a solid support; (iii) comparing the effect of the presenceof said test molecule on the amount of said cleaved complex to theamount of said cleaved complex in the absence of said test molecule; and(iv) identifying the test molecule as a botulinum neurotoxin inhibitorif the presence of said test molecule decreases the relative amount ofcleaved complex produced in said cells as compared to the amount ofcleaved complex produced in said cells in the absence of said testmolecule; wherein said botulinum neurotoxin in step (i) is delivered tosaid cells by means selected from administering a botulinum neurotoxinto said cells and expressing said botulinum neurotoxin in said cells bya recombinant vector; and wherein said botulinum neurotoxin is fromabout 80% to 100% homologous to an amino acid sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14, wherein said neurotoxin iscapable of cleaving a specific cleavage site of said neurotoxin'sendogenous substrate.